The human pathogen Yersinia pestis, as well as its close relatives Yersinia pseudotuberculosis and Yersinia enterocolitica, employ a virulence plasmid-encoded type Ill secretion pathway to escape phagocytic killing during host infection. Type III secretion provides for Y. pestis multiplication and spread within lymphoid tissues as well as for the pathogenesis of acute lethal infections. The Yersinia type III pathway transports 13 polypeptides either into the extra-cellular medium (YopBDR and LcrV) or into the cytosol of host cells (YopEHMNOPT and LcrQ). During infection, Yersinia receive host-specific environmental cues that are transduced to relieve repression of the type III pathway. Several regulatory mechanisms of gene expression and type III transport have emerged. Moreover, the type III machinery represents a unique secretory system that recognizes its substrates via signals encoded in yop mRNA. The genes and mechanisms that are required for type III substrate recognition and regulation of gene expression or transport are still unknown. The virulence plasmid of Y. pestis encodes components of a type III secretion machine, its transport substrates and several regulatory factors. Preliminary studies show that the chromosome of yersiniae encodes additional genes involved in type III secretion. The purpose of this proposal is to exploit the Y. pestis genome sequence, the Y. pestis growth-restricted phenotype and the availability of mini-Mu transposable elements for random insertional mutagenesis, thereby isolating all Y. pestis genes that are required for type III secretion. Identified genes will be characterized further to reveal the mechanisms of substrate recognition, regulation of type III transport and pathogenesis of plague infections. Together these studies are also aimed at identifying molecular targets for drug therapies, an important area of research to aid in the defense against biological warfare and bioterrorist deployment of the select agent Y. pestis. [unreadable] [unreadable]